This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is Superoxide Dismutase-4 (SOD-4).
There is a very strong thermodynamic driving force for the reactions between oxygen and biochemical compounds in the body such as proteins, carbohydrates, lipids and nucleic acids. If such reactions go to completion, water, carbon dioxide and a number of waste products are formed as end products with the release of large amounts of energy. Oxidation of biological compounds is the source of energy of living organisms. Such reactions occur spontaneously but are very slow due to reaction barriers. These barriers are overcome by enzymes in intermediary metabolism, and the final reaction with oxygen takes place in the mitochondria, where the oxygen is reduced by four electrons to water without the liberation of any intermediate products. The reaction is accomplished by cytochrome oxidase complex in the electron transport chain and the energy is bound by the formation of ATP.
However, the direct four step reduction of oxygen to water is unique, and when oxygen reacts spontaneously or is catalyzed by enzymes it is forced to react one step at a time. A series of reactive and toxic intermediates are formed, namely the superoxide radical (O2xe2x88x92), hydrogen peroxide (H2O2), and the hydroxyl radical (OHxe2x88x92).
Two of these, O2xe2x88x92 and OHxe2x88x92, have single unpaired electrons and are therefore called free radicals. A few percent of the oxygen consumption in the body has been estimated to lead to the formation of the toxic reduction intermediates. The toxic affects of oxygen are mainly ascribable to the actions of these intermediates.
Oxygen in itself reacts slowly with most biochemical compounds. The toxic reactions are in general initiated by processes giving rise to oxygen radicals, which in themselves cause direct damage to biochemical compounds or start chain reactions involving oxygen.
Some compounds react spontaneously with oxygen, i.e., they autoxidize. Virtually all autoxidations result in the formation of toxic oxygen reduction intermediates. Autoxidation of adrenalin, pyrogallol and several other compounds lead to the formation of the superoxide radical. When ionizing radiation passes through an aqueous solution containing oxygen, the superoxide radical is the radical found in the highest concentration. The toxic oxygen reduction products so formed are of fundamental importance for the killing ability of the cells, but may also lead to damage in the surrounding tissue.
Hydrogen peroxide is always formed when superoxide is formed by way of the dismutation reaction. Most oxidases in the body directly reduce oxygen to hydrogen peroxide.
Organisms living in the presence of oxygen have been forced to develop a number of protective mechanisms against the toxic oxygen reduction metabolites. The protective factors include superoxide dismutases (SOD) which dismutate the superoxide radical and are found in relatively constant amounts in mammalian cells and tissue. The best known of these enzymes is CuZnSOD which is a dimer with a molecular weight of 33,000 containing two copper and two zinc atoms. CuZnSOD is found in the cytosol and in the intermembrane space of the mitochondria. MnSOD is a tetramer with a molecular weight of 85,000 containing four Mn atoms, and is mainly located in the mitochondrial matrix. Until recently the extra cellular fluids were assumed to lack SOD activity. However U.S. Pat. No. 5,248,603 recently disclosed the presence of a superoxide dismutase in extracellular fluids (e.g., blood plasma, lymph, synovial fluid and cerebrospinal fluid) which was termed EC-SOD.
Crystallographic structures of recombinant human CuZnSOD have been determined, refined and analyzed at 2.5 A resolution for wild-type and a designed thermal stable double-mutant enzyme (Cys-6----Ala, Cys-111----Ser). There is a helix dipole interaction with a Zn site, and 14 residues form two or more structurally conserved side-chain to main-chain hydrogen bonds that appear critical to active-site architecture, loop confirmation and the increased stability resulting from the Cys-111----Ser mutation. Parge, H. E. et al, Proc. Natl. Acad. Sci. U.S.A., 89:6109-13 (1992).
Mutations in the CuZnSOD gene occur in patients with the fatal neurodegenerative disorder familial amyotrophic lateral sclerosis. Screening of the CuZnSOD coding region revealed that the mutation Ala 4 to Val in exon 1 was the most frequent one, mutations were identified in exons 2, 4 and 5 but not in the active site region formed by exon 3. Thus, defective CuZnSOD is linked to motor neuron death and carries implications for understanding and possible treatment of familial amyotrophic lateral sclerosis. The polypeptide of the present invention, SOD-4, is structurally and functionally related to CuZnSOD.
Japanese Patent No. 4327541 discloses a therapeutic drug for immuno-reactions with organs after transplantation containing the active substance of human CuZnSOD obtained by gene recombination.
Japanese Patent No. 4312533 discloses a composition for treating cerebral ischaemia which comprises recombinant CuZn human SOD and inhibits delayed nerve necrosis accompanying ischaemia.
Japanese Patent No. 4248984 discloses a superoxide dismutase derivative which has a longer half-life in blood than SOD and therefore helps treat various diseases.
European Patent No. 499621 discloses a method for purifying recombinant CuZnSOD and a method for increasing the yield of the B isoform analog of this polypeptide.
Japanese Patent No. 2156884 discloses a 153 amino acid polypeptide having human superoxide dismutase properties and a DNA sequence encoding such polypeptide, a DNA sequence expressed by the nucleic acid sequence and production of the polypeptide by culture of host cells.
Japanese Patent No. 63313581 discloses a pharmacologically active modified superoxide dismutase which is obtained by reacting SOD with a compound containing an amino or carboxyl group.
Japanese Patent No. 63077822 discloses an agent for improving the function of organs which uses a human SOD-like polypeptide as the active substance.
In accordance with one aspect of the present invention, there is provided a novel mature polypeptide which is SOD-4, as well as fragments, analogs and derivatives thereof. The polypeptide of the present invention is of human origin.
In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such polypeptides.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides for therapeutic purposes, for example, for treating inflammatory pathologies, ulcers, arrhythmia, ischaemia, oedema, paraquat intoxication, rheumatoid arthritis and osteoarthritis, reducing reperfusion injuries and decreasing blood pressure.
In accordance with yet a further aspect of the present invention, there is provided an antibody against such polypeptides. These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.